This invention relates to an immunoassay method for detecting specific antibodies from biological fluid test samples using known antigens in a simple, reliable and efficient manner. More particularly, the present invention provides a single step procedure for detecting antibodies and reliably quantitating the concentration thereof without requiring a high level of skill by the test administrator. A field diagnostic kit for veterinary and human medical purposes or a professional office laboratory kit is included in the scope of the present invention.
Immunoassay or immunocytochemical assay methods have developed greatly over the past two decades. The many assay methods currently available to detect antibodies present in clinical specimens can generally be divided into two groups technically: (a) multi-step procedures, and (b) agglutination or precipitation type reactions. The multi-step procedures include many published assay methods using markers such as enzymes, radioactive labels, fluorescent labels, colloidal labels, or complement fixation. These methods require several steps to reach a definitive and lasting result, such as binding of antibodies to the target antigens, rinsing off unbound antibodies, and then detecting the remaining bound antibodies through the use of one of the labels. Subsequent preserving or mounting steps are also necessary in some cases. The agglutination and precipitation type reactions are dependent on and, therefore, limited to the detection of antibodies which have agglutinating or precipitating characteristics, are difficult to apply to more than one test antigen in a single procedure, and are relatively insensitive. Published reports of solid phase immunoassays using labeled colloidal gold or other labeled suspended particles to detect antibodies follow the long established, tedious and time consuming protocol and assay configuration that requires lengthy incubations of test sample to bind the antibodies, followed by a series of rinsing steps and at least one additional lengthy incubation with the colloidal label, see Moeremans et al, J. Immunological Methods, Vol. 74, 1984, pp. 353-360; Moeremans et al, "Sensitive Colloidal Metal (Gold or Silver) Staining of Protein Blots on Nitrocellulose Membranes", Analytical Biochemistry, Vol. 145, 1985, pp. 315-321; Surek and Latzko, "Visualization of Antigenic Proteins Blotted Onto Nitrocellulose Using the Immuno-Gold-Staining (IGS) Method", Biochemical and Biophysical Research Communications, Vol. 121, No. 1., 1984, pp. 284-289; and Yau-Heiu Hsu, "Immunogold for Detection of Antigen on Nitrocellulose Paper", Analytical Biochemistry, Vol. 142, 1984, pp. 221-225, to detect the antigen bound antibodies.
One visualization method for an immunoassay according to the general methodology of blot overlay assays using colloidal metal particles is shown in European Patent No. 158,746, published Oct. 23, 1985. This patent teaches the use of nitrocellulose paper as a blotting medium which is spotted with either tubulin or calmodulin, the remaining binding sites were quenched by incubating with bovine serum albumin (BSA), then the antigen containing nitrocellulose strips were incubated, respectively, with anti-tubulin and anti-calmodulin serum, followed by washing three times with BSA-Tris. Then the washed strips were incubated with the colloidal gold labeling material and again washed with BSA-Tris before evaluation. A comparison with an immobilized peroxidase incubation showed similar positivity, expressed differently however. Although the foregoing procedure employs a novel blot labeling technique, e.g., colloidal metal particle sols, the method itself is quite similar to the previously known enzyme labeled immunoassay method. This is seen from Example 1 in which the same method, using the two different labeling techniques, is run in side-by-side comparison and with similar results.
While the assay methods of the prior art provide accurate results, it is always desirable to achieve those results faster, with simpler and more reliable methods of increased sensitivity and at lower cost of both expensive biological materials and the analyst's labor. Therefore, it is an object of the present invention to provide an immuno-assay method which is simpler than the prior art procedures. A further object of the present invention is to provide an immunoassay method which is faster and allows very sensitive detection without instrumentation. Another object of the present invention is the provision of an immunoassay kit for field testing human and veterinary species or for use in a professional office laboratory. Other objects and advantages of the present invention will be apparent from the following description thereof.